Anti β actin antibody clone ac 15

Western blot analysis concentrated on proteins involved in tumor cell differentiation and migration processes and included E- and N-cadherin, vimentin and cytokeratin (CK) 8/18 expression. Tumor cell lysates were applied to a polyacrylamide gel and run for 90 min at 100 V. The concentration of acrylamide ranged from 3% to 12%, depending on the evaluated protein. The proteins were then transferred to nitrocellulose membranes for 1 h at 100 V and blocked with non-fat dry milk (1 h). Overnight incubation was finally carried out with monoclonal antibodies directed against E-cadherin, N-cadherin, vimentin, and CK 8/18 (all were from Cell Signaling, Leiden, The Netherlands). As secondary antibodies, HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG were used at a 1:5000 dilution (both were from Upstate Biotechnology, Lake Placid, NY, USA). Proteins were visualized by the ECL detection reagent (Amersham/GE Healthcare, München, Germany) and analyzed with the Fusion FX7 system (Peqlab, Erlangen, Germany). Internal controls were carried out with an anti-β-actin antibody (clone AC-15; Sigma-Aldrich). To quantify the intensity of the protein bands, the protein/β-actin intensity ratio was quantified using GIMP 2.8 software.

In total, 5 × 10⁵ cells of each cell line were seeded and cultivated overnight, treated with cetuximab-protamine (60 nM) coupled to the indicated siRNAs at 1:10 molar ratio once a day for 3 days, harvested, lysed in RIPA buffer and cleared by ultrasonification and centrifugation. Xenograft tumours were homogenised as 10% w/v in RIPA buffer using an ultraturrax, cleared by ultrasonification and centrifugation. Western blot analysis was performed using standard protocols with the following antibodies: anti-KRAS (ab55391, Abcam, Cambridge, UK), and anti β-Actin antibody (Clone AC-15, Sigma Aldrich).

The mouse monoclonal antibody 9B11 reactive against the c-Myc epitope (EQKLISEEDL) and the rabbit polyclonal antiserum reactive against human PLCγ₂ (sc-407) were obtained from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. The rabbit polyclonal antiserum reactive against human Rac2 (sc-96) was purchased from Santa Cruz Biotechnology. The anti-β-actin antibody (clone AC-15) and the anti-Rac1 antibody (clone 23A8) were obtained from Sigma and Merck Millipore, respectively. The Rac inhibitor EHT 1864 and its inactive analog EHT 4063 were synthesized as described previously [41 ]. Human epidermal growth factor (EGF) (E9644) was from Sigma. ProGreen baculovirus vector DNA (A1) was purchased from AB Vector.

Vero cells were inoculated with 17syn⁺ or 17-37 (MOI 1). At 24 HPI, cells were washed with PBS and lysed with 3x lysis buffer (30% stacking gel buffer [0.5 M Tris with 0.4% SDS, pH 6.8], 30% glycerol, 6.5% SDS powder, 9M urea, 100mm DTT and bromophenol blue) diluted to 1x with PBS. Samples were run on a 4–20% mini-PROTEAN TGX precast protein gel (Biorad, cat. # 4561093) and transferred onto an Amersham Protran nitrocellulose membrane (Sigma-Aldrich, cat. # GE10600002). Blocking was done in 5% non-fat milk. The gC and β-actin (control) proteins were detected using HSV-1 anti gC antibody clone M701139 (Fitzgerald, cat. # 10-H25A, batch 912, mouse monoclonal, 1:1000 dilution), anti-β-actin antibody clone AC-15 (Sigma, cat. # A5441, lot no. 127M866V, mouse monoclonal, 1:1000 dilution), and anti-mouse IgG, human ads-HRP antibody (Southern Biotech, cat. # 1030–05, lot no. K3515-T566G, goat, 1:1000 dilution).

APPswe lysates from both SST-scFv8D3
treated and PBS treated groups, as well as WT controls, were mixed
with LDS sample buffer (Thermo Scientific, Waltham, MA, USA) and Bolt
sample reducing agent (Thermo Scientific, Waltham, MA, USA) and loaded
onto Bolt 4–12% Bis-tris plus gels (Thermo Scientific, Waltham,
MA, USA). Gels were run at 80 V for 1–2 h followed by transfer
onto PVDF-membranes (Merck, Darmstadt, Germany). Membranes were blocked
with 5% dry milk in TBS-Tween for 1 h at RT, followed by overnight
staining at 4 °C with primary antibodies anti-syntaxin antibody
(ab41453, Abcam, Cambridge, United Kingdom) and anti-β-actin
antibody, clone AC-15 (Sigma-Aldrich, Stockholm, Sweden). The following
day, membranes were washed with TBS-Tween buffer and incubated with
goat antimouse IgG Alexa 680 (A21057, Life Technologies, Waltham,
MA, USA) and donkey antirabbit IgG Alexa 800 (A32808, Invitrogen,
Waltham, MA, USA) secondary antibodies for 1 h at RT. After they were
washed three times with TBS-Tween buffer, signals were developed using
the LI-COR odyssey machine (LI-COR biosciences, Homburg, Germany).
Signal intensity was measured using Image Studio software (version
5.2.5). The measured signals from syntaxin were normalized against
the loading control (β-actin).